PHYTOCHEMICAL INVESTIGATION ON SOME
SPECIES FROM THE GENERA ELETTARIOPSIS AND
ETLINGERA
YASODHA SIVASOTHY
UNIVERSITI SAINS MALAYSIA
2008
PHYTOCHEMICAL INVESTIGATION ON SOME SPECIES FROM
THE GENERA ELETTARIOPSIS AND ETLINGERA
by
YASODHA SIVASOTHY
Thesis submitted in fulfillment of the
requirements for the Degree of
Doctor of Philosophy
MAY 2008
ACKNOWLEDGEMENT
Firstly, I would like to take this opportunity to thank my supervisor, Assoc. Prof. Dr. Wong
Keng Chong for his advice and guidance. I am also thankful to my co-supervisor, Prof. Dr.
Boey Peng Lim for his encouragement and helpful suggestions throughout the course of this study.
Next, I would like to acknowledge the Dean of the Institute of Graduate Studies (IPS) for giving me a chance to pursue my postgraduate studies in USM. Special thanks to the Dean of the School of Chemical Sciences, Prof. Dr. Wan Ahmad Kamil Mahmood for providing me with the assistance and facilities which ensured the success of my research.
I would also like to thank Institut Pengajian Siswazah for awarding me with the Graduate
Assistant Scheme which covered my allowance and my tuition fee.
I am very grateful to Datuk Lim Chong Keat from FRIM and Mr. Baharuddin Sulaiman from the School of Biological Sciences, USM for providing me with the plant materials and in the identification of them for my research. I would also like to thank Mr. Shanmugam from the School of Biological Sciences, USM for helping me prepare the voucher specimens for certain of those species.
I would like to forward my appreciation to the technical and laboratory staffs of the School of Chemical Sciences, in particular, Mr. Chow Cheng Por, Mr. Clement D’Silva, Mr. Tan
Chin Tong, Mr. Megat Hasnul, Mr. Yee Chin Leng, Mr. Chee Sai Gnow, Mr. Aw Yeong and Mr. Mohd. Fahmi Mohd. Yusoff for their assistance during the duration of this study.
ii
I would like to acknowledge Mr. Khoo Kay Hock and Mr. Hilman from the Centre for Drug
Research, USM for their help with the GC-MS analyses.
Special thanks to Madam Wong Lai Kwai and Madam Han Yan Hui from the Department of Chemistry, National University of Singapore for their help with the DP-MS and the NMR analyses.
I would also like to thank my colleagues and friends for their cooperation and moral support
throughout this project.
Finally, I would like to convey my deepest gratitude to my parents for their love and encouragement.
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TABLE OF CONTENTS
Page
Acknowledgement ii
Table of Contents iv
List of Tables viii
List of Figures xi
List of Schemes xvi
List of Abbreviations xvii
List of Appendices xix
Abstrak xxii
Abstract xxiv
CHAPTER ONE: INTRODUCTION 1
1.1 Natural Products Chemistry 1
1.2 The Zingiberaceae Family 1
1.2.1 The Origin of the Word ‘Ginger’ 1
1.2.2 Habitat 1
1.2.3 Distribution 2
1.2.4 Use and Commercial Importance 2
1.2.5 The Genus Elettariopsis 4
1.2.6 The Genus Etlingera 9
1.2.7 Previous Phytochemical Investigations 12
1.2.7.1 The Genus Elettariopsis 12
1.2.7.2 The Genus Etlingera 12
1.3 Research Objectives 13
1.3.1 Part 1 13
1.3.2 Part 2 14
iv
CHAPTER TWO: MATERIALS AND METHODS 15
2.1 Collection of the Plant Materials 15
2.2 Chemicals and Reagents 15
2.3 Isolation and Analysis of Essential Oils 16
2.3.1 Isolation of Essential Oils 16
2.3.2 Chromatographic Analysis of the Essential Oils 18
2.3.2.1 Gas Chromatography 18
2.3.2.2 Gas Chromatography-Mass Spectrometry 20
2.3.3 Identification of the Essential Oil Components 20
2.3.4 Determination of Oil Yield 20
2.4 Isolation and Characterization of the Non-Volatile Constituents in the 21
Leaves, Fruits, Rhizomes and Roots of Etlingera littoralis
2.4.1 Extraction Procedure 21
2.4.1.1 Leaves 21
2.4.1.2 Fruits 21
2.4.1.3 Rhizomes and Roots 21
2.4.2 Separation Techniques 22
2.4.2.1 Thin Layer Chromatography 22
2.4.2.2 Column Chromatography 22
2.4.2.3 Preparative Thin Layer Chromatography 22
2.4.3 Isolation and Purification 23
2.4.3.1 Leaves 23
2.4.3.1.1 Petroleum Ether Extract 23
2.4.3.2 Fruits 24
2.4.3.2.1 Ethyl Acetate Extract 24
2.4.3.2.2 Petroleum Ether Extract 24
2.4.3.3 Rhizomes and Roots 25
v
2.4.3.3.1 Ethyl Acetate Extract 25
2.4.3.3.2 Petroleum Ether Extract 27
2.4.4 Structure Elucidation of Isolated Compounds 28
2.4.4.1 Melting Point 28
2.4.4.2 Infrared Spectroscopy 28
2.4.4.3 Gas Chromatography-Mass Spectrometry 28
2.4.4.4 Direct Probe-Mass Spectrometry 29
2.4.4.5 Nuclear Magnetic Resonance Spectroscopy 29
2.4.4.6 Ultra-Violet Spectroscopy (UV) of F1 29
2.4.4.6.1 Preparation of the Stock Solution of F1 30
2.4.4.6.2 Preparation of the Shift Reagent Stock Solutions and Solids 30
2.4.4.6.3 Steps in the UV Spectral Analysis of F1 30
2.4.4.7 Ultra-Violet Spectroscopy (UV) of L1, F2 and R4 31
2.4.4.8 Optical Rotation 31
CHAPTER THREE: RESULTS AND DISCUSSION 32
3.1 Essential Oil Analysis 32
3.1.1 Percentage Yield of Essential Oil 32
3.1.2 Chemical Composition of the Various Essential Oils 32
3.1.2.1 Essential Oils of Elettariopsis smithiae 33
3.1.2.2 Essential Oils of Elettariopsis rugosa 37
3.1.2.3 Essential Oils of Elettariopsis elan 41
3.1.2.4 Essential Oils of Elettariopsis slahmong 45
3.1.2.5 Essential Oils of Elettariopsis triloba 48
3.1.2.6 Essential Oils of Elettariopsis curtisii 52
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3.1.2.7 Significance of the Essential Oils in the Chemotaxonomic 56
Studies of the Genus Elettariopsis
3.1.2.8 Essential Oils of Etlingera littoralis 64
3.1.2.9 Essential Oils of Etlingera elatior and Etlingera elatior var. 68
Thai queen
3.2 Non-volatile Constituents Isolated from the Leaves, Fruits, Rhizomes and 75
Roots of Etlingera littoralis
3.2.1 Leaves 75
3.2.1.1 L1 75
3.2.1.2 L2 87
3.2.2 Fruits 107
3.2.2.1 F1 107
3.2.2.2 F2 122
3.2.3 Rhizomes and Roots 138
3.2.3.1 R1 138
3.2.3.2 R2 153
3.2.3.3 R3 168
3.2.3.4 R4 178
3.2.3.5 R5 185
CHAPTER FOUR: CONCLUSION 204
REFERENCES 206
APPENDICES 216
LIST OF PUBLICATIONS 234
vii
LIST OF TABLES
Page
Table 2.1: Weight of Plant Material and Volume of Distilled Water Used for 17 Hydrodistillation
Table 3.1: Percentage Yields of the Essential Oils of the Various Ginger Species 32
Table 3.2: Constituents of the Essential Oils of the Leaves and Roots of 34 Elettariopsis smithiae
Table 3.3: Percentages of the Various Chemical Classes in the Essential Oils of 36 the Leaves and Roots of Elettariopsis smithiae
Table 3.4: Constituents of the Essential Oils of the Leaves and Roots of 38 Elettariopsis rugosa
Table 3.5: Percentages of the Various Chemical Classes in the Essential Oils of 40 the Leaves and Roots of Elettariopsis rugosa
Table 3.6: Constituents of the Essential Oils of the Leaves and Roots of 42 Elettariopsis elan
Table 3.7: Percentages of the Various Chemical Classes in the Essential Oils of 44 the Leaves and Roots of Elettariopsis elan
Table 3.8: Constituents of the Essential Oils of the Leaves and Roots of 46 Elettariopsis slahmong
Table 3.9: Percentages of the Various Chemical Classes in the Essential Oils of 47 the Leaves and Roots of Elettariopsis slahmong
Table 3.10: Constituents of the Essential Oils of the Leaves and Roots of 49 Elettariopsis triloba
Table 3.11: Percentages of the Various Chemical Classes in the Essential Oils of 51 the Leaves and Roots of Elettariopsis triloba
Table 3.12: Constituents of the Essential Oils of the Leaves and Roots of 53 Elettariopsis curtisii
Table 3.13: Percentages of the Various Chemical Classes in the Essential Oils of 55 the Leaves and Roots of Elettariopsis curtisii
Table 3.14: Composition of the Essential Oils Isolated from Six Elettariopsis 59 Species
Table 3.15: Constituents of the Essential Oils of the Leaves, Rhizomes and 65 Roots of Etlingera littoralis
Table 3.16: Percentages of the Various Chemical Classes in the Essential Oils of 67 the Leaves, Rhizomes and Roots of Etlingera littoralis
viii
Table 3.17: Constituents of the Essential Oils of the Leaves, Rhizomes and 71 Roots of Etlingera elatior and Etlingera elatior var. Thai queen
Table 3.18: Percentages of the Various Chemical Classes in the Essential Oils of 73 the Leaves, Rhizomes and Roots of Etlingera elatior
Table 3.19: Percentages of the Various Chemical Classes in the Essential Oils of 74 the Leaves, Rhizomes and Roots of Etlingera elatior var. Thai queen
1 1 1 Table 3.20: H NMR (δppm) Assignments and H- H COSY Correlations of L1 in 82 CDCl3, 400 MHz
13 1 13 1 13 Table 3.21: C NMR (δppm) Assignments and H- C HMQC and H- C HMBC 83 Correlations of L1 in CDCl3, 75 MHz
1 1 1 Table 3.22: H NMR (δppm) Assignments and H- H COSY Correlations of L2 in 98 CDCl3, 400 MHz
13 1 13 1 13 Table 3.23: C NMR (δppm) Assignments and H- C HMQC and H- C HMBC 99 Correlations of L2 in CDCl3, 75 MHz
Table 3.24: Important 1H-1H NOESY and 1H-1H ROESY Correlations of L2 in 105 CDCl3, 500 MHz
Table 3.25: UV analysis of F1 in MeOH and the Effects of the Various Shift 107 Reagents
1 1 1 Table 3.26: H NMR (δppm) Assignments and H- H COSY Correlations of F1 in 120 CD3OD, 400 MHz
13 1 13 1 13 Table 3.27: C NMR (δppm) Assignments and H- C HMQC and H- C HMBC 120 Correlations of F1 in CD3OD, 75 MHz
Table 3.28: Optical Rotations and Melting Points of Naturally Occurring 121 Catechins (Dictionary of Natural Products, 1996)
Table 3.29: IR Spectral Data of F2 132
1 1 1 Table 3.30: H NMR (δppm) Assignments and H- H COSY Correlations of F2 in 133 CDCl3, 400 MHz
13 1 13 1 13 Table 3.31: C NMR (δppm) Assignments and H- C HMQC and H- C 134 HMBC Correlations of F2 in CDCl3, 75 MHz
Table 3.32: IR Spectral Data of R1 147
1 1 1 Table 3.33: H NMR (δppm) Assignments and H- H COSY Correlations of R1 in 148 CDCl3, 400 MHz
13 1 13 1 13 Table 3.34: C NMR (δppm) Assignments and H- C HMQC and H- C 149 HMBC Correlations of R1 in CDCl3, 75 MHz
Table 3.35: IR Spectral Data of R2 162
ix
1 1 1 Table 3.36: H NMR (δppm) Assignments and H- H COSY Correlations of R2 162 in CDCl3, 400 MHz
13 1 13 1 13 Table 3.37: C NMR (δppm) Assignments and H- C HMQC and H- C HMBC 164 Correlations of R2 in CDCl3, 75 MHz
1 1 1 Table 3.38: H NMR (δppm) Assignments and H- H COSY Correlations of R3 in 175 CDCl3, 400 MHz
13 1 13 1 13 Table 3.39: C NMR (δppm) Assignments and H- C HMQC and H- C HMBC 175 Correlations of R3 in CDCl3, 75 MHz
1 Table 3.40: H NMR (δppm) Assignments of R4 in CDCl3, 400 MHz 184
13 Table 3.41: C NMR (δppm) Assignments of R4 in CDCl3, 75 MHz 184
Table 3.42: IR Spectral Data of R5 196
1 1 1 Table 3.43: H NMR (δppm) Assignments and H- H COSY Correlations of R5 in 198 CDCl3, 400 MHz
Table 3.44: Pyridine-Induced Solvent Shifts of the Proton Signals Characteristic 198 of R5, 400 MHz
13 1 13 1 13 Table 3.45: C NMR (δppm) Assignments and H- C HMQC and H- C HMBC 199 Correlations of R5 in CDCl3, 75 MHz
x
LIST OF FIGURES
Page
Fig. 1.1: Elettariopsis triloba (Gagnep.) Loesen. (Kam, 1982) 7
Fig. 1.2: Elettariopsis curtisii Bak. (Kam, 1982) 8
Fig. 1.3: Elettariopsis smithiae Kam (Kam, 1982) 8
Fig. 1.4: Elettariopsis rugosa (Kam) C.K. Lim (Kam, 1982) 9
Fig. 1.5: Etlingera elatior (Jack) R.M. Smith (Henderson, 1954) 11
Fig. 2.1: Hydrodistillation Apparatus 17
Fig. 2.2: Gas Chromatogram Checking the Purity of R1 26
Fig. 2.3: Gas Chromatogram Checking the Purity of R2 26
Fig. 3.1: Mass Spectrum of L1 76
Fig. 3.2: UV Spectrum of L1 in Ethanol 77
Fig. 3.3: FTIR Spectrum of L1 78
Fig. 3.4: 1H NMR Spectrum of L1 79
Fig. 3.5: 13C NMR Spectrum of L1 80
Fig. 3.6: DEPT 135 NMR Spectrum of L1 81
Fig. 3.7: DEPT 90 NMR Spectrum of L1 81
Fig. 3.8: Structure of α-tocopherol (Matsuo and Urano, 1976) 82
Fig. 3.9: 1H-1H COSY NMR Spectrum of L1 84
Fig. 3.10: 1H-13C HMQC NMR Spectrum of L1 85
Fig. 3.11: 1H-13C HMBC NMR Spectrum of L1 86
Fig. 3.12: Basic skeleton of a pimarane compound 87
Fig. 3.13: Mass Spectrum of L2 88
Fig. 3.14: FTIR Spectrum of L2 89
Fig. 3.15: 13C NMR Spectrum of L2 90
Fig. 3.16: DEPT 135 NMR Spectrum of L2 91
Fig. 3.17: DEPT 90 NMR Spectrum of L2 91
Fig. 3.18: 1H NMR Spectrum of L2 92
xi
Fig. 3.19: Basic skeleton of a sandaracopimaradiene compound 93
Fig. 3.20: 1H-13C HMBC NMR Spectrum of L2 96
Fig. 3.21: Acetoxyl group at C4 of a sandaracopimaradiene skeleton 97
Fig. 3.22: Structure of sandaracopimara-8(14),15-diene-18-yl formate (Seca et 97 al., 2008)
Fig. 3.23: 1H-1H COSY NMR Spectrum of L2 100
Fig. 3.24: 1H-13C HMQC NMR Spectrum of L2 101
Fig. 3.25: 1H-1H NOESY NMR Spectrum of L2 103
Fig. 3.26: 1H-1H ROESY NMR Spectrum of L2 104
Fig. 3.27: Structure of L2 105
Fig. 3.28: The NOE enhancement results observed in the NOESY and 106 ROESY spectra of L2
Fig. 3.29: UV Spectrum of F1 in MeOH 107
Fig. 3.30: Mass Spectra of F1 108
Fig. 3.31: FTIR Spectrum of F1 109
Fig. 3.32: 13C NMR Spectrum of F1 110
Fig. 3.33: DEPT 135 NMR Spectrum of F1 111
Fig. 3.34: DEPT 90 NMR Spectrum of F1 111
Fig. 3.35: UV Spectrum of F1 (MeOH) After the Addition of NaOAc 113
Fig. 3.36: UV Spectrum of F1 (MeOH) After the Addition of NaOMe 114
Fig. 3.37: UV Spectrum of F1 (MeOH) After the Addition of AlCl3 and HCl 114
Fig. 3.38: UV Spectrum of F1 (MeOH) After the Addition of NaOAc and 114 H3BO3
Fig. 3.39: 1H NMR Spectrum of F1 115
Fig. 3.40: 1H-1H COSY NMR Spectrum of F1 116
Fig. 3.41: 1H-13C HMQC NMR Spectrum of F1 118
Fig. 3.42: 1H-13C HMBC NMR Spectrum of F1 119
Fig. 3.43: Structure of catechin (Lai et al., 1985) 120
Fig. 3.44: Structure of epi-catechin (Lai et al., 1985) 121
xii
Fig. 3.45: Structure of F1 122
Fig. 3.46: UV Spectrum of F2 in Ethanol 123
Fig. 3.47: Mass Spectrum of F2 124
Fig. 3.48: 13C NMR Spectrum of F2 125
Fig. 3.49: DEPT 135 NMR Spectrum of F2 126
Fig. 3.50: DEPT 90 NMR Spectrum of F2 126
Fig. 3.51: 1H NMR Spectrum of F2 127
Fig. 3.52: FTIR Spectrum of F2 129
Fig. 3.53: 1H-13C HMBC NMR Spectrum of F2 130
Fig. 3.54: 1H-1H COSY NMR Spectrum of F2 131
Fig. 3.55: Structure of (E)-labda-8(17),12-diene-15,16-dial (Itokawa et al., 132 1988)
Fig. 3.56: 1H-13C HMQC NMR Spectrum of F2 135
Fig. 3.57: Structure of F2 136
Fig. 3.58: Mass Spectrum of R1 139
Fig. 3.59: FTIR Spectrum of R1 140
Fig. 3.60: 13C NMR Spectrum of R1 141
Fig. 3.61: DEPT 135 NMR Spectrum of R1 142
Fig. 3.62: DEPT 90 NMR Spectrum of R1 142
Fig. 3.63: 1H NMR Spectrum of R1 143
Fig. 3.64: An equatorial hydroxyl group at C3 of a sandaracopimaradiene 145 skeleton
Fig. 3.65: 1H-13C HMBC NMR Spectrum of R1 146
Fig. 3.66: Structure of sandaracopimara-8(14),15-diene-3β-ol (Misra and Dev, 1986) 147
Fig. 3.67: Structure of ent-8(14),15-sandaracopimaradiene-2α,18-diol 148 (Chamacho et al., 2001)
Fig. 3.68: 1H-1H COSY NMR Spectrum of R1 150
Fig. 3.69: 1H-13C HMQC NMR Spectrum of R1 151
Fig. 3.70: Mass Spectrum of R2 155
xiii
Fig. 3.71: FTIR Spectrum of R2 156
Fig. 3.72: 13C NMR Spectrum of R2 157
Fig. 3.73: DEPT 135 NMR Spectrum of R2 158
Fig. 3.74: DEPT 90 NMR Spectrum of R2 158
Fig. 3.75: 1H NMR Spectrum of R2 159
Fig. 3.76: Basic skeleton of an isopimaradiene compound 160
Fig. 3.77: Hydroxyl group at C3 of an isopimaradiene skeleton 161
Fig. 3.78: 1H-13C HMBC NMR Spectrum of R2 163
Fig. 3.79: Structure of isopimara-7,15–diene-3β-ol (Block et al., 2004) 164
Fig. 3.80: 1H-1H COSY NMR Spectrum of R2 165
Fig. 3.81: 1H-13C HMQC NMR Spectrum of R2 166
Fig. 3.82: Mass Spectrum of R3 169
Fig. 3.83: FTIR Spectrum of R3 170
Fig. 3.84: 13C NMR Spectrum of R3 171
Fig. 3.85: DEPT 135 NMR Spectrum of R3 172
Fig. 3.86: DEPT 90 NMR Spectrum of R3 172
Fig. 3.87: 1H NMR Spectrum of R3 173
Fig. 3.88: 1H-13C HMBC NMR Spectrum of R3 174
Fig. 3.89: Structure of R3 175
Fig. 3.90: 1H-1H COSY NMR Spectrum of R3 176
Fig. 3.91: 1H-13C HMQC NMR Spectrum of R3 177
Fig. 3.92: UV Spectrum of R4 in Ethanol 178
Fig. 3.93: Mass Spectrum of R4 179
Fig. 3.94: FTIR Spectrum of R4 180
Fig. 3.95: 13C NMR Spectrum of R4 181
Fig. 3.96: DEPT 135 NMR Spectrum of R4 182
Fig. 3.97: DEPT 90 NMR Spectrum of R4 182
Fig. 3.98: 1H NMR Spectrum of R4 183
xiv
Fig. 3.99: Structure of R4 184
Fig. 3.100: Mass Spectrum of R5 186
Fig. 3.101: FTIR Spectrum of R5 187
Fig. 3.102: 13C NMR Spectrum of R5 188
Fig. 3.103: DEPT 135 NMR Spectrum of R5 189
Fig. 3.104: DEPT 90 NMR Spectrum of R5 189
Fig. 3.105: 1H NMR Spectrum of R5 190
Fig. 3.106: Basic skeleton of a 15-isopimarene (15-sandaracopimarene) 191 compound
Fig. 3.107: Hydroxymethyl group at C4 of a 15-isopimarane skeleton 192
Fig. 3.108: 1H-13C HMBC NMR Spectrum of R5 193
Fig. 3.109: Hydroxyl group at C8 of a 15-isopimarane skeleton 194
Fig. 3.110: Structure of 15-isopimarene-8,19-diol (Stierle et al., 1988) 196
1 Fig. 3.111: H NMR Spectrum of R5 in pyridine-d5 197
Fig. 3.112: 1H-1H COSY NMR Spectrum of R5 200
Fig. 3.113: 1H-13C HMQC NMR Spectrum of R5 201
xv
LIST OF SCHEMES
Page
Scheme 3.1 75
Scheme 3.2 94
Scheme 3.3 94
Scheme 3.4 112
Scheme 3.5 123
Scheme 3.6 144
Scheme 3.7 144
Scheme 3.8 154
Scheme 3.9 160
Scheme 3.10 191
Scheme 3.11 195
Scheme 3.12 195
Scheme 3.13 195
xvi
LIST OF ABBREVIATIONS
GC: Gas Chromatography
GC-MS: Gas Chromatography-Mass Spectrometry
FID: Flame Ionization Detector
RI: Retention Index
Rt: Retention Time
TLC: Thin Layer Chromatography
UV: Ultra-Violet Spectroscopy
Prep-TLC: Preparative Thin Layer Chromatography
Prep-GC: Preparative Gas Chromatography
TCD: Thermal Conductivity Detector
FT-IR: Fourier Transform Infrared
DP-MS: Direct Probe-Mass Spectrometry
EI: Electron Ionization
NMR: Nuclear Magnetic Resonance
DEPT: Distortionless Enhancement by Polarization Transfer
COSY: Correlated Spectroscopy
HMQC: Heteronuclear Multiple Quantum Correlation
HMBC: Heteronuclear Multiple Bond Correlation
NOESY: Nuclear Overhauser Enhancement Spectroscopy
ROESY: Rotating frame Overhauser Effect Spectroscopy
NOE: Nuclear Overhauser Effect ppm: parts per million s: singlet d: doublet t: triplet q: quartet
xvii
m: multiplet dd: double doublets br: broad eV: electron volts amu: atomic mass unit ax: axial eq: equatorial s: strong w: weak
xviii
LIST OF APPENDICES
Page
Appendix A1: Isolation of L1 and L2 216
Appendix A2: Isolation of F1 218
Appendix A3: Isolation of F2 219
Appendix A4: Isolation of R1, R2, R3 and R4 220
Appendix A5: Isolation of R5 221
Appendix B1: Gas Chromatogram of Elettariopsis smithiae Leaf Oil on the 222 SPB-1 Column
Appendix B2: Gas Chromatogram of Elettariopsis smithiae Leaf Oil on the 222 Suplecowax 10 Column
Appendix B3: Gas Chromatogram of Elettariopsis smithiae Root Oil on the 222 SPB-1 Column
Appendix B4: Gas Chromatogram of Elettariopsis smithiae Root Oil on the 223 Supelcowax 10 Column
Appendix C1: Gas Chromatogram of Elettariopsis rugosa Leaf Oil on the 223 SPB-1 Column
Appendix C2: Gas Chromatogram of Elettariopsis rugosa Leaf Oil on the 223 Supelcowax 10 Column
Appendix C3: Gas Chromatogram of Elettariopsis rugosa Root Oil on the 224 SPB-1Column
Appendix C4: Gas Chromatogram of Elettariopsis rugosa Root Oil on the 224 Supelcowax 10 Column
Appendix D1: Gas Chromatogram of Elettariopsis elan Leaf Oil on the 224 SPB-1 Column
Appendix D2: Gas Chromatogram of Elettariopsis elan Leaf Oil on the 225 Supelcowax 10 Column
Appendix D3: Gas Chromatogram of Elettariopsis elan Root Oil on the 225 SPB-1 Column
Appendix D4: Gas Chromatogram of Elettariopsis elan Root Oil on the 225 Supelcowax 10 Column
Appendix E1: Gas Chromatogram of Elettariopsis slahmong Leaf Oil on the 226 SPB-1 Column
Appendix E2: Gas Chromatogram of Elettariopsis slahmong Leaf Oil on the 226 Supelcowax 10 Column
xix
Appendix E3: Gas Chromatogram of Elettariopsis slahmong Root Oil on the 226 SPB-1 Column
Appendix E4: Gas Chromatogram of Elettariopsis slahmong Root Oil on the 227 Supelcowax 10 Column
Appendix F1: Gas Chromatogram of Elettariopsis triloba Leaf Oil on the 227 SPB-1 Column
Appendix F2: Gas Chromatogram of Elettariopsis trloba Leaf Oil on the 227 Supelcowax 10 Column
Appendix F3: Gas Chromatogram of Elettariopsis triloba Root Oil on the 228 SPB-1 Column
Appendix F4: Gas Chromatogram of Elettariopsis triloba Root Oil on the 228 Supelcowax 10 Column
Appendix G1: Gas Chromatogram of Elettariopsis curtisii Leaf Oil on the 228 SPB-1 Column
Appendix G2: Gas Chromatogram of Elettariopsis curtisii Leaf Oil on the 229 Supelcowax 10 Column
Appendix G3: Gas Chromatogram of Elettariopsis curtisii Root Oil on the 229 SPB-1 Column
Appendix G4: Gas Chromatogram of Elettariopsis curtisii Root Oil on the 229 Supelcowax 10 Column
Appendix H1: Gas Chromatogram of Etlingera littoralis Leaf Oil on the 230 SPB-1 Column
Appendix H2: Gas Chromatogram of Etlingera littoralis Leaf Oil on the 230 Supelcowax 10 Column
Appendix H3: Gas Chromatogram of Etlingera littoralis Root Oil on the 230 SPB-1 Column
Appendix H4: Gas Chromatogram of Etlingera littoralis Root Oil on the 231 Supelcowax 10 Column
Appendix I1: Gas Chromatogram of Etlingera elatior Leaf Oil on the 231 SPB-1 Column
Appendix I2: Gas Chromatogram of Etlingera elatior Leaf Oil on the 231 Supelcowax 10 Column
Appendix I3: Gas Chromatogram of Etlingera elatior Root Oil on 232 the SPB-1Column
Appendix I4: Gas Chromatogram of Etlingera elatior Root Oil on the 232 Supelcowax 10 Column
xx
Appendix J1: Gas Chromatogram of Etlingera elatior var. Thai queen Leaf Oil 232 on the SPB-1Column
Appendix J2: Gas Chromatogram of Etlingera elatior var. Thai queen Leaf Oil 233 on the Supelcowax 10 Column
Appendix J3: Gas Chromatogram of Etlingera elatior var. Thai queen Root Oil 233 on the SPB-1Column
Appendix J4: Gas Chromatogram of Etlingera elatior var. Thai queen Root Oil 233 on the Supelcowax 10 Column
xxi
KAJIAN FITOKIMIA KE ATAS BEBERAPA SPESIES DARIPADA
GENERA ELETTARIOPSIS DAN ETLINGERA
ABSTRAK
Minyak pati bahagian daun dan akar enam spesies Elettariopsis dan tiga spesies Etlingera yang diperolehi dari Malaysia telah dipencilkan melalui kaedah penghidrosulingan dan dianalisiskan dengan kaedah GC kapilari dan GC-MS dengan menggunakan dua kolum dengan kekutuban yang berbeza. Kedua-dua minyak pati Elettariopsis smithiae didominasi oleh monoterpena, dengan komponen utama geranial (38.1%) dan neral (29.1%) dalam minyak pati daun, dan kamfena (22.9%) dan α-fenchil asetat (15.7%) dalam minyak pati
akar. Minyak pati daun E. rugosa mempunyai kandungan tinggi seskuiterpena, terutamanya
spatulenol (29.5%), sementara kebanyakan komponen dalam minyak pati akarnya adalah
monoterpena dengan β-felandrena (26.2%) dan kamfena (15.2%) dua komponen utama yang
hadir dalam kandungan paling tinggi. Kedua-dua minyak pati daun dan akar E. elan
mempunyai kandungan monoterpena yang tinggi dengan geraniol (71.6%) sebagai
komponen major dalam minyak pati daun manakala kamfena (28.6%), α-fenchil asetat
(8.6%) dan α-felandrena (8.4%) dalam minyak pati akar. Komponen bukan terpena
membentuk sebahagian besar minyak pati daun dan akar E. slahmong dengan aldehid
alifatik, terutamanya trans-2-oktenal (46.3% and 8.1%, masing-masing) dan trans-2-dekenal
(36.8% and 79.4%, masing-masing) membentuk bahagian utama. Monoterpena didapati
mendominasi profil minyak pati daun dan akar E. triloba, dengan β-pinena (31.2%), neral
(12.3%), geranil asetat (11.3%) dan geranial (10.7%) merupakan komponen utama dalam
minyak pati daun sementara kamfena (29.3%) dan α-felandrena (10.4%) mencirikan minyak
pati akarnya. Minyak pati daun dan akar E. curtisii mempunyai kandungan tinggi
monoterpena dengan β-pinena (42.7% and 29.6%, masing-masing) and β-felandrena (19.9% and 17.6%, masing-masing) dua komponen utama dalam kedua-dua minyak pati tersebut.
xxii
Minyak pati daun Etlingera littoralis dicirikan oleh kehadiran trans-metil isoeugenol
(37.7%), β-pinena (30.4%) dan β-felandrena (8.6%) manakala trans-metil isoeugenol
(58.1%) dan sandarakopimara-8(14),15-diena-3β-ol (9.1%) didapati dominan dalam minyak pati rizom dan akarnya. Komponen utama dalam minyak pati daun E. elatior adalah mirsena
(13.5%), α-humulena (11.8%), β-kariofilena (10.7%), dodekanol (9.9%) dan α-pinena
(8.5%) sementara minyak pati rizom dan akarnya mengandungi kuantiti monoterpena yang tinggi dan dicirikan dengan kehadiran kamfena (18.0%), β-pinena (16.9%), bornil asetat
(9.2%) dan α-pinena (8.6%). Minyak pati daun E. elatior var. Thai queen dicirikan oleh
komponen bukan terpena manakala minyak pati rizom dan akarnya mempunyai kandungan
monoterpena yang tinggi. α-Pinena (24.4%), dodekanol (18.9%) dan dodekanal (15.9%) merupakan komponen utama dalam daun tetapi rizom dan akarnya dicirikan dengan kehadiran kamfena (15.1%), dodekanol (12.9%), bornil asetat (10.7%) and dodekanal
(10.6%).
Bahagian rizom, akar, buah dan daun E. littoralis diekstrakan secara berasingan dengan
menggunakan pelarut petroleum eter diikuti etil asetat. Setiap ekstrak ditulenkan melalui
kaedah kromatografi kolum dengan menggunakan gel silika atau Sephadex LH-20, kaedah
TLC penyediaan atau kaedah GC penyediaan untuk memberi sembilan sebatian. Ketulenan
setiap sebatian diuji sama ada dengan TLC atau GC dan strukturnya ditentukan dengan
kaedah-kaedah spektroskopi seperti IR, GC-MS, DP-MS, 1D NMR, 2D NMR dan UV. Data
putaran optik spesifik bagi sebatian-sebatian tertentu diperolehi untuk membantu dalam
mengenalpasti isomer optik yang sebenar. Sandarakopimara-8(14),15-diena-3β-ol,
isopimara-7,15–diena-3β-ol, 15-isopimarena-8β,19-diol, trans-metil isoeugenol dan metil vanilin telah diidentifikasikan dalam bahagian rizom dan akar, ent-katecin dan ent-(E)- labda-8(17),12-diena-15,16-dial pula dipencilkan daripada buah manakala α-tokoferol dan
19β–asetoksisandarakopimara-8(14),15-diena, sejenis diterpena baru daripada kelas pimarana, didapati hadir dalam daun.
xxiii
PHYTOCHEMICAL INVESTIGATION ON SOME SPECIES FROM THE
GENERA ELETTARIOPSIS AND ETLINGERA
ABSTRACT
The volatile oils of the aerial and underground parts of six Malaysian Elettariopsis and three
Etlingera species were isolated by hydrodistillation and analysed by capillary GC and GC-
MS, using two columns of different polarity. The oils of Elettariopsis smithiae were dominated by monoterpenoids, the major components being geranial (38.1%) and neral
(29.1%) in the leaf oil, and camphene (22.9%) and α-fenchyl acetate (15.7%) in the oil from the roots. The leaf oil of E. rugosa contained high levels of sesquiterpenoids, particularly spathulenol (29.5%), while the root oil contained mainly monoterpenoids, with β- phellandrene (26.2%) and camphene (15.2%) being clearly the two most abundant components. Both the leaf and root oils of E. elan were overwhelmingly monoterpenoid in character, with geraniol (71.6%) accounting for the major part of the leaf oil, while camphene (28.6%), α-fenchyl acetate (8.6%) and α-phellandrene (8.4%) characterized the root oil. Non-terpenoids constituted most of the leaf and root oils of E. slahmong, with
aliphatic aldehydes, particularly trans-2-octenal (46.3% and 8.1%, respectively) and trans-
2-decenal (36.8% and 79.4%, respectively), accounting for the major part. Monoterpenoids dominated the profile of the leaf and root oils of E. triloba, with β-pinene (31.2%), neral
(12.3%), geranyl acetate (11.3%) and geranial (10.7%) the principal contributors in the leaf oil, while camphene (29.3%) and α-phellandrene (10.4%) characterized the root oil. The
leaf and root oils of E. curtisii were principally monoterpenoid, with β-pinene (42.7% and
29.6%, respectively) and β-phellandrene (19.9% and 17.6%, respectively) being the two most prominent components in both.
xxiv
The leaf oil of Etlingera littoralis was characterized by the occurrence of trans-methyl
isoeugenol (37.7%), β-pinene (30.4%) and β-phellandrene (8.6%), while in the rhizome and root oil, trans-methyl isoeugenol (58.1%) and sandaracopimara-8(14),15-diene-3β-ol (9.1%) predominated. Regarding the leaf oil of E. elatior, the major components were myrcene
(13.5%), α-humulene (11.8%), β-caryophyllene (10.7%), dodecanol (9.9%) and α-pinene
(8.5%). The rhizome and root oil, however, was dominated by monoterpenoids, with camphene (18.0%), β-pinene (16.9%), bornyl acetate (9.2%) and α-pinene (8.6%) clearly the most abundant. Non-terpenoids were predominant in the leaf oil of E. elatior var. Thai queen while the rhizome and root oil was dominated by monoterpenoids. α-Pinene (24.4%),
dodecanol (18.9%) and dodecanal (15.9%) were the major constituents present in the leaves,
while in the rhizome and root oil, camphene (15.1%), dodecanol (12.9%), bornyl acetate
(10.7%) and dodecanal (10.6%) predominated.
The air-dried rhizomes, roots, fruits and leaves of E. littoralis were separately fractionated
by solvent extraction using petroleum ether followed by ethyl acetate. Each extract was
further fractionated by repeated column chromatography over either silica gel or Sephadex
LH-20, preparative TLC or preparative GC, giving nine isolated compounds. The purity of
each of the compounds was examined using either TLC or GC, and its structure elucidated
by spectroscopic techniques such as IR, GC-MS, DP-MS, 1D NMR, 2D NMR and UV.
Specific optical rotation data were also obtained for certain of these compounds to aid in the
identification of the exact optical isomer. Sandaracopimara-8(14),15-diene-3β-ol, isopimara-
7,15–diene-3β-ol, 15-isopimarene-8β,19-diol, trans-methyl isoeugenol and methyl vanillin were identified from the rhizomes and roots, ent-catechin and ent-(E)-labda-8(17),12-diene-
15,16-dial from the fruits, α-tocopherol and a new pimarane type diterpene, 19β– acetoxysandaracopimara-8(14),15-diene, from the leaves.
xxv
CHAPTER ONE
INTRODUCTION
1.1 Natural Products Chemistry
Natural Products Chemistry is a field focused on the discovery of new compounds with biological activities which may be of medical importance and which are produced by plants, animals or other organisms. Extracts are assayed for biological activity and the active fractions are purified to find the active compound(s). Structural characterization of the compounds by spectroscopic and related analytical methods complete the preliminary investigations which may lead to new therapeutic drugs.
1.2 The Zingiberaceae Family
1.2.1 The Origin of the Word ‘Ginger’
The word ‘ginger’ truly refers to the edible ginger of commerce known in the Malay language as halia and botanically as Zingiber officinale Roscoe, while ‘gingers’ is a general term for members of the ginger family. The name Zingiber probably originated from the
Arabic word zanjabil and later the Sanskrit word singabera (meaning horn-root), which gave rise to the classical Greek name zingiberi and finally zingiber in Latin. Botanically,
Zingiber gives its name to the whole ginger family, Zingiberaceae (Larsen et al., 1999).
1.2.2 Habitat
Gingers thrive in a wide range of habitats from riverine to limestone rocks and from the lowlands to the upper montane regions. Most gingers are terrestrial, growing naturally in damp, humid, shady areas with good light but several native species can tolerate the full exposure of the sun.
Gingers are generally abundant in lowland to hill forests, notably between 200 m and 500 m above sea level. Gingers are less profuse in higher altitudes and rather scarce on very high
1
mountains. On some isolated islands which are relatively dry, the diversity of gingers is quite low and sometimes they are even absent. Some species inhabit secondary forests, open places such as along road-sides, forest gaps, riverbanks and swampy vegetation. The climate of Peninsular Malaysia allows continuous growth of the rhizomes throughout the year although more vigorous growth may be apparent during the rainy season (Larsen et al.,
1999).
1.2.3 Distribution
These are plants of tropical and subtropical regions distributed mainly in Asia. The
Zingiberaceae comprises about 1200 species of which about 1000 occur in Tropical Asia.
By far, the richest area is the Malesian region, a floristically distinct region that includes
Malaysia, Indonesia, Brunei, Singapore, the Philippines and Papua New Guinea, with 24 genera and about 600 species. With the present knowledge, there are about 18 genera with more than 160 species of Zingiberaceae in Peninsular Malaysia. These include Alpinia,
Amomum, Boesenbergia, Camptandra, Curcuma, Elettaria, Elettariopsis, Etlingera,
Geocharis, Geostachys, Globba, Haniffia, Hedychium, Hornstedtia, Kaempferia,
Plagiostachys, Scaphochlamys and Zingiber. Many of these are rare and very local in distribution and consequently highly vulnerable to endangerment (Larsen et al., 1999).
1.2.4 Use and Commercial Importance
The main gingers of use come from the genera Alpinia, Amomum, Curcuma and Zingiber, and, to a lesser extent, Boesenbergia, Kaempferia, Elettaria, Elettariopsis, Etlingera and
Hedychium. At least 20 or more ginger species have been cultivated for their use as spices, condiments, flavours, fresh vegetables, medicine, ornamentals and cut flowers. The presence of essential oils in many Zingiberaceae species have made some species important since the time of the ancient Greek (Larsen et al., 1999).
2
One of the earliest uses was as spices. Zingiber officinale (halia in Malay) is one of the best
and oldest known spices of the Zingiberaceae. Until today, it is still in demand as one of the
ingredients in food, bakeries (ginger bread, biscuits) confectionaries, beverages (ginger
beer) and traditional medicine. The old rhizomes are used fresh in flavouring while its
young rhizomes are eaten raw or pickled as a relish. Turmeric, known as Curcuma
domestica Val. in Peninsular Malaysia and Curcuma longa L. in India and other Asian
countries is popular (after ginger) as a spice used in curries. It is also used as a food
flavouring and in ancient times it was even exploited as a dye. The broad aromatic leaves of
Curcuma domestica are used for wrapping fish before steaming or baking. Turmeric has a
long list of uses ranging from spice, flavour, traditional medicine and in cultural beliefs and
rites. Alpinia galanga (L.) Sw. is a minor spice in some western countries. In Malaysia, its
rhizomes are called lengkuas and much used in the spicy meat dish called rendang. In
Malaysia, the leaves of Kaempferia galanga L. (called cekur) is familiar in perut ikan, a
favourite local dish. The young rhizomes of Curcuma mangga Val. & Van Zijp,
Boesenbergia rotunda (L.) Mansf. and Zingiber zerumbet Smith, and young inflorescence of
Curcuma domestica and Alpinia galanga are also consumed as fresh vegetables or ulam (a term equivalent to salad) by the village folk (Ibrahim, 1992).
Many studies and surveys have shown that at least more than ten cultivated species of
Zingiberaceae have been frequently used in traditional medicine. Many of the medicinal gingers are used in traditional cures which are apparently associated with women-related ailments or illness, e.g. post-partum medicines for women during confinement. Ginger species including Curcuma zedoria (Berg.) Rosc. (temu kuning in Malay), Curcuma mangga
(temu pauh), Curcuma aeruginosa Roxb. (temu hitam) and Zingiber montanum (Koenig)
Theilade (bonglai) are used in food preparations for women in confinement after birth. The
rhizomes of Zingiber officinale and Curcuma domestica are frequently used as a carminative
for relieving flatulence. The latter is also used as an anti-spasmodic in diarrhoea. Similarly,
Curcuma xanthorhiza Roxb. (temu lawak), Zingiber ottensi Val. (lempoyang hitam or
3
bonglai hitam) and Zingiber zerumbet (lempoyang) have roles in herbal medicine (Khaw,
1995). Besides these, Globba species are occasionally used in traditional medicine (Ibrahim,
1995).
1.2.5 The Genus Elettariopsis
Baker (1892) founded the genus Elettariopsis, describing three species, two of which were
found on Penang Hill: E. serpentina Bak. and E. curtisii Bak. collected by Curtis. The third
Baker taxon, E. exserta was collected by Scortechini from ‘Goping, Straits Settlements’ in
June 1885. Ridley (1907) listed seven species including the ones in Baker, adding E.
latiflora Ridl. (which he first found in Singapore prior to 1899), E. pubescens Ridl. (based
on Amomum biflorum Jack), E. cyanescens [now Haniffia cyanescens (Ridl.) Holtt.] and E.
longituba [now Elettaria longituba (Ridl.) Holtt.]. Holttum (1950) dealt with Ridley’s E.
pubescens, reverting it to Amomum biflorum Jack. He also considered E. latiflora Ridl. as a
synonym of E. curtisii.
Despite Scortechini’s clear diagnosis of E. exserta, it is still remained unknown and
uncertain to Holttum who resorted to regard it as a larger form of E. curtisii. According to
Lim (2003), E. curtisii is clearly not to be confused with E. latiflora and E. exserta. He is
convinced that E. curtisii is endemic to Penang Island while E. exserta and E. latiflora are
found mainly in the south of the Peninsula, in Johor and Singapore.
Based on Lim’s (2003) findings, there appear to be three forms of E. triloba, one scentless
and the other two strongly aromatic but different. According to this authority, the ‘true’ E.
triloba has yet to be ascertained in the wild within Peninsular Malaysia. The two aromatic
forms which resembled E. triloba have been elevated in rank by Lim to two new species and
are quite wide spread throughout Peninsular Thailand and Malaysia. One of these, E.
slahmong, possesses a stink bug scent resembling that exuded by insects such as
Catacanthus incarnatus and Tantao ocellatus. The largest population of this species has
4
been observed in Tioman. The other species, which has a very strong and sweet lemon scent, is recognized by Lim as E. elan. However, Holttum recognized E. triloba (Gagnep.)
Loesen., which is a Vietnamese species, as extant in the Peninsula, ascribing it to a local
species which seemed similar. According to Kam (1982), E. triloba is found in Perak,
Selangor, Pahang and Johor. Holttum’s assertion that the crushed leaves of both E. curtisii
and E. triloba exude an unpleasant pungent odour somewhat similar to that emitted by
various kinds of bugs, can be corrected based on the fact the E. triloba which he was
referring to was actually E. slahmong, one of the two new species identified by Lim (2003).
Kam (1982) recognized two new species and a new variety, E. burttiana and E. smithiae and
its variety rugosa. E. burttiana is mainly confined to central Perak (Lim, 2003). E. smithiae
and its variety rugosa differ in the shape and width of the lamina and also its colour sheen.
Both have a distinct lemon scent but the variety rugosa has a trace of stink bug odour in it.
Lim (2003) elevated the variety to the status of a new species and named it E. rugosa.
In advance of an ongoing revision of the genus Elettariopsis in Peninsular Malaysia, nine species have been confirmed by Lim (2003): E. elan C.K. Lim, E. slahmong
C.K. Lim, E. triloba (Gagnep.) Loesen., E. curtisii Bak., E. smithiae Kam, E. rugosa (Kam)
C.K. Lim, E. exserta (Scort.) Bak., E. latiflora Ridl. and E. burttiana Kam.
E. elan
This is a highly aromatic herb with a lemon scent. It is 60 to 90 cm in height, arising from creeping rhizomes. The leaves, 2-5 in number, consist of leafless sheaths, which form distinct pseudostems 5 x 8 mm to 25 cm at close or distant intervals from the creeping rhizomes. Their laminas are lanceolate or ovate, with a dimension of 35 x 5-8 cm, and are dark green in colour. The inflorescence emerges from the basal shoots, with penducle 1-5 cm forming a cincinnus of 7-10 flowers exerting from the bracts. Flowers with labellum are
5
of typical coloration, white with red median stripe flanked by yellow streaks. The fruit is globular, green to reddish brown in colour, six-ridged with a diameter of 2 cm (Lim, 2003).
E. slahmong
This is an aromatic herb with all parts of the plant emitting a stink-bug odour. It is 50 to 145 cm in height, arising from creeping rhizomes. The leaves, 2-6 in number form pseudostems.
Their laminas are ovate or lanceolate, with a dimension of 60 cm x 13.5 cm (70 x 8.5 cm), and are light green in colour. The inflorescence emerges from the shoot or extended base shoot and clusters in a cincinnus. The bracts are cream or light green in colour. The fruit is globular, six-ridged with a diameter of 2-2.5 cm. The herb is delectable to the orang asli and much sought after by them to be used in native cuisine for cooking fish. In Southern
Thailand, the leaves of E. slahmong are eaten as a salad (Lim, 2003).
E. triloba
The rhizomes are slender, wide-creeping and horizontal. They bear leaf-shoots at intervals of 8-15 cm. The plants are not tuffed and the roots are not tuberous. The leaf-shoots consist of 1-5 leaves which grow on pseudostems of tightly clasping leaf-sheaths which grow up to
35 cm tall. The laminas of the leaves are lanceolate with a dimension of 30 x 5-8 cm. Both surfaces of the leaves are glabrous. The inflorescence emerges from the base of the leaf- shoot, consisting of 4-8 bracts in a compact head. All the bracts and calyx are suffused pink, each subtending 1-2 flowers. The labellum is three-lobed, the middle lobe being cream with broad yellow median band bordered by a red stripe on either side towards the throat (Kam,
1982).
6
Fig. 1.1: Elettariopsis triloba (Gagnep.) Loesen. (Kam, 1982)
E. curtisii
This is a rather small plant with long-creeping slender rhizomes. The leaf shoots arise from them at widely spaced intervals of 6-20 cm. Each shoot consists of 1-5 leaves. The leaves are glabrous and erect, with loosely clasping sheaths of length 5-30 cm and release a strange stink bug odour when they are bruised. The leaves are used as flavours and traditional medicines by the orang asli. The laminas are elliptic, with dimensions of 24 x 4 – 68 x 10 cm, tapering to both ends. The inflorescence is long and slender, emerging from the base of the leaf-shoot, just below ground surface. The bracts are small with white calyx. The lobes are white, pale yellow with an orange centre and radiating crimson lines. The fruit is a globular capsule, with a diameter of 3 cm. It is shallowly ridged; pink speckled with dark red dots and contains white seeds (Kam, 1982; Keng et al., 1998).
7
Fig. 1.2: Elettariopsis curtisii Bak. (Kam, 1982)
E. smithiae
This is an aromatic herb which emits a lemon scent when its leaves are crushed. The leafy shoot has a distinct pseudostem with 3-8 or sometimes 9 greyish green floppy leaves which are arranged in two ranks. The flowers have white, yellow and red patterns and are produced on a long branching inflorescence. The fruit is a pale brown spherical capsule. It is slightly ridged, 3 cm in diameter, and formed along the leaf litter (Larsen et al., 1999).
Fig. 1.3: Elettariopsis smithiae Kam (Kam, 1982)
8
E. rugosa
This herb is strongly aromatic. The scent of the crushed leaves incorporates a perceptible and a variable odour of the stink bug (less pungent than in E. slahmong) over the lemon smell. The leaves of this herb are elliptical (or ovate) and rugose (Lim, 2003).
Fig. 1.4: Elettariopsis rugosa (Kam) C.K. Lim (Kam, 1982)
1.2.6 The Genus Etlingera
In ‘The Zingiberaceae of the Malay Peninsula’, Holttum (1950) listed four species under
Phaeomeria Lindely. He later realized that Lindely’s Phaeomeria (1836) was invalid,
despite Schumann’s later adoption and (in his 1974 paper) endorsed the generic change to
Nicolaia, which was named by Horaninow in 1862 to honour the late Emperor of Russia
(Lim, 2000).
Prompted by a suggestion by Holttum, Burtt and Smith (1986) proposed the combination of
the three genera Achasma Griff., Geanthus Val. (not found in Malaysia) and Nicolaia
Horan. under Etlingera, which was first used in 1972 by Giseke (Lim, 2000).
Following the change of genus name, Smith (1986) embarked on a sweeping exercise in
combinations, covering some 60 species over the whole regional distribution range, and
9
largely on the basis of assiduous herbarium research, much of it by interpretation of literature and without being able to see type specimens (Lim, 2000).
E. elatior
E. elatior (Jack) R.M. Smith or the Torch Ginger is synonymous with Phaeomeria speciosa
(Blume) Merr., Phaeomeria magnifica (Roscoe) K. Schum. or Nicolaia speciosa (Blume)
Horan. A native to Asia, it is one of the most beautiful of all tropical flowering plants. It is a robust, coarse and large herb growing in clumps of 3-6 m tall. The leaf blades are lanceolate, with a dimension of 85 x 15 cm, and are arranged in two alternate rows upon the stem. The lower surface of the leaf is often purplish when young while the top surface is glossy green. Its inflorescence comes out of the ground instead of the terminal spike. The flower heads are broadly cone-shaped, 7-10 cm long, subtended by large crimson or pink
(with white edge) bracts and seated on a stalk of about 1 m long. The corolla is pink, lip is crimson with a narrow white or yellow margin, and the anther is red. The fruit, hairy and green to reddish in colour, is a globose, with a diameter of 2.5 cm and contains many black seeds (Henderson, 1954; Keng et al., 1998). The plant is grown in tropical locations for both
the extravagant flowers and as food. In Malaysia, where it is called kantan, the flower shoot is a compulsory ingredient of laksa asam, a favourite noodle dish in Peninsular Malaysia.
The herb is also used in local dishes such as nasi kerabu, nasi ulam and tom-yam (Larsen et al., 1999).
10
Fig. 1.5: Etlingera elatior (Jack) R.M. Smith (Henderson, 1954)
E. elatior var. Thai queen
This herb is a variety of E. elatior. It is morphologically similar to E. elatior, the only difference is the colour of the inflorescence which is white.
E. littoralis
The synonyms of E. littoralis (Koenig) Giseke include Achasma megalocheilos Griff. and
Hornstedtia megalocheilos Ridl. The herb has stout stems which are 4-5 m tall. The leaves which are glabrous are broadly oblong, 0.5-1 m long, with a short stalk of length 1-1.5 cm.
It has a spike like-inflorescence 5-8 cm long partly embedded in the ground. The bracts are ovate or oblong, papery, pale pink with a crimson tip, subtending 4-12 flowers which open together. The calyx is pink and tubular, 7-8 cm long. The corolla is of the same length with pink lobes. The lip is red with yellow or orange margins. The fruit is a capsule globose, 3 cm in diameter (Keng et al., 1998; Sha Ren Shu, 2000).
11
1.2.7 Previous Phytochemical Investigations
1.2.7.1 The Genus Elettariopsis
Two variants of E. triloba, designated as variant 1 and 2, were investigated for their volatile components by Mustafa et al. (1996). These workers found that the two variants showed differences in their volatile constituents. Variation was also detected when different parts
(leaf, rhizome, root) of the plant were compared (Larsen et al., 1999). Examples of the major components in the leaves of variant 1 included neral (12.5%), geranial (16.1%), 2,7- dimethyl-2,6-octadien-1-ol (14.1%) and geranyl isobutyrate (10.5%), while the rhizome oil contained limonene (11.3%) and 2-carene (9.1%). In contrast, the major components in the leaf oil of variant 2 included β-caryophyllene (20.2%) and eremophilene (22.7%). While the rhizome oil contained 1,8-cineole (15.1%) and borneol (9.0%), the roots were found to yield camphene (14.1%) and β-caryophyllene (18.9%).
1.2.7.2 The Genus Etlingera
Some volatile components of E. elatior have been identified in three previous investigations.
The first, by Wong et al. (1993), reported on the essential oil composition obtained by hydrodistillation of the young flower shoots of the herb growing in Malaysia. They positively identified dodecanol (33.2%), dodecanal (17.2%), α-pinene (13.7%) and
dodecanoic acid (7.4%) as the major components.
In a later study, Zoghbi and Andrade (2005) investigated the volatile constituents in the oil
of the inflorescence and inflorescence axis of this herb cultivated in the state of Para, Brazil,
using GC and GC-MS. The inflorescence and inflorescence axis oils yielded dodecanol
(42.5% and 34.6%), dodecanal (14.5% and 21.5%) and α-pinene (22.2% and 6.3%) as the
main constituents, respectively.
12
Recently, Jaafar et al. (2007) analysed the essential oils isolated from different parts (leaves, stems, flowers and rhizomes) of Malaysian E. elatior, using GC-MS. The leaf oil was found to contain β-pinene (19.7%), β-caryophyllene (15.4%) and trans-β-farnesene (27.1%) as the
major compounds whereas the stem oil was largely dominated by 1,1-dodecanediol
diacetate (34.3%) and trans-5-dodecene (27.0%). The oils of the flowers and rhizomes
contained 1,1-dodecanediol diacetate (24.4% and 40.4%, respectively) and cyclododecane
(47.3% and 34.5%, respectively) as the major compounds.
Phytochemical studies on the rhizomes of E. elatior (Mohamad et al., 2005) afforded two
new compounds, 1,7-bis(4-hydroxyphenyl)-2,4,6-heptatrienone and 16-hydroxylabda-
8(17),11,13-trien-15,16-olide, along with demethoxycurcumin, 1,7-bis(4-hydroxyphenyl)-1,
4,6-heptatrien-3-one, stigmast-4-en-3-one, stigmast-4-ene-3,6-dione, stigmast-4-en-6β-ol-3- one, and 5α,8α-epidioxyergosta-6,22-dien-3β–ol.
1.3 Research Objectives
1.3.1 Part 1
Since the treatments by Holttum (1950) and Kam (1982), recognition of some of the members of the Elettariopsis has become confused. The ongoing revision by Lim (2003) to unravel species which have been wrongly identified was based on morphology and scent.
However, utilizing scent alone can be misleading, and classifying species based on this method is insufficient and unreliable. The discernibly different odour of the essential oils suggests the possible use of their principal constituents as taxonomic markers for species identification (Jantan et al., 1994; Salguerio, 1993). It will be interesting to see if the
essential oil composition can be used as a complementary tool in the taxonomy of species of
this genus.
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Objectives:
1. To identify the essential oil constituents in the leaves and roots of E. smithiae, E.
rugosa, E. elan, E. slahmong, E. triloba and E. curtisii using capillary GC and GC-
MS techniques.
2. To carry out a comparative study between the essential oil composition of the leaves
and roots of each species mentioned in (1.).
3. To carry out a comparative study of the essential oil composition of E. smithiae, E.
rugosa, E. elan, E. slahmong, E. triloba and E. curtisii in an attempt to develop a
taxonomic guide for species identification.
4. To confirm whether the two new species identified by Lim (2003), E. elan and E.
slahmong, are identical or not to the two variants of E. triloba investigated by
Mustafa et al. (1996).
1.3.2 Part 2
Objectives:
1. To identify the essential oil constituents in the leaves, rhizomes and roots E. elatior,
E. elatior var. Thai queen and E. littoralis using capillary GC and GC-MS
techniques.
2. To carry out a comparative study between the essential oil composition of the
leaves, rhizomes and roots of of each species or variety mentioned in (1.).
3. To isolate and elucidate the structure of the major non-volatile constituents, in
particular, the phenolics and terpenoids present in the leaves, fruits, rhizomes and
roots of E. littoralis, using various spectroscopic techniques.
14
CHAPTER TWO
MATERIALS AND METHODS
2.1 Collection of the Plant Materials
The leaves and roots of Elettariopsis elan, E. slahmong, E. smithiae and E. rugosa were collected from a home garden in Penang. Voucher specimens have been deposited with the herbarium of the Forestry Research Institute Malaysia (FRIM): E. elan C.K. Lim L6306
(KEP); E. slahmong C.K. Lim L6101 (KEP); E. smithiae Kam L3953 (KEP); E. rugosa
(Kam) C.K. Lim L6197 (KEP). E. triloba was collected in the campus of Universiti Sains
Malaysia, and E. curtisii from the Ayer Itam Dam, Penang. Voucher specimens of these two species have been deposited with the herbarium of the School of Biological Sciences,
U.S.M. (10950 and 10946, respectively).
Etlingera elatior was collected from the Penang Botanical Garden, E. elatior var. Thai queen from a home garden in Penang, and E. littoralis from the Ayer Itam Dam, Penang.
Voucher specimens of all these plants have been deposited with the herbarium of the School of Biological Sciences, U.S.M. (10615, 10765 and 10947, respectively).
2.2 Chemicals and Reagents
1. *Acetone, AR grade (Systerm, Malaysia)
2. Aluminium Chloride, AlCl3.6H2O (Merck, Germany)
3. Boric Acid (Sigma-Aldrich, USA)
4. *Chloroform, AR grade (R&M Chemicals, UK)
5. Chloroform-d, with 0.03 v/v % TMS, 99.8 atom % D, stabilized with silver
foil (Acros Organics, USA)
6. Diethyl Ether, AR grade (Lab-Scan, Thailand)
7. *Ethyl Acetate, AR grade (Fisher Scientific, UK)
8. *Ethanol 95%, AR grade (Systerm, Malaysia)
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9. Ferric Chloride, FeCl3.6H2O (Merck, Germany)
10. Hydrochloric Acid 37% (Fischer Chemicals, UK)
11. *Methanol, AR grade (Systerm, Malaysia)
12. Methyl-d3 alcohol-d, with 0.03 v/v % TMS, 99.8 atom % D (Acros Organics, USA)
13. *Pentane, AR grade (Merck, Germany)
14. *Petroleum Ether 60-80 oC, AR grade (Lab-Scan, Thailand)
15. Sephadex LH-20 (Sigma-Aldrich, USA)
16. Silica Gel 60 for column chromatography, (0.040-0.063 mm) (230-400 mesh
ASTM) (Merck, Germany)
17. Anhydrous Sodium Acetate (R&M Chemicals, UK)
18. Sulphuric Acid 95-98% (Systerm, Malaysia)
19. TLC Aluminium Sheets, Silica Gel 60 F254, 20 cm x 20 cm (Merck, Germany)
20. Silica Gel 60 F254, pre-coated glass plates 20 cm x 20 cm x 0.5 mm (Merck,
Germany)
* Solvents were distilled prior to use
2.3 Isolation and Analysis of Essential Oils
2.3.1 Isolation of Essential Oils
Fresh intact leaves, chopped roots (for plants from the genus Elettariopsis) and rhizomes and roots (for plants from the genus Etlingera) were separately subjected to hydrodistillation to isolate the essential oils, carrying out by using an all-glass apparatus similar to that described in the British Pharmacopoaeia (1993) (Fig. 2.1).
The plant material (15-80 g), cleaned by rinsing under running tap water followed by distilled water to remove soil and dirt, was placed in a 0.5 L round bottom flask which was filled with distilled water (300-400 ml) to a level such that the entire plant material was immersed (Table 2.1).
16
condenser
inlet A
pentane
distilled water
round bottom flask plant material + distilled water heating mantle
Fig. 2.1: Hydrodistillation Apparatus
Table 2.1: Weight of Plant Material and Volume of Distilled Water Used for Hydrodistillation
Species Plant material Weight (g) Volume of distilled water (ml) Elettariopsis smithiae leaves 40 400 roots 50 400 Elettariopsis rugosa leaves 70 400 roots 80 400 Elettariopsis elan leaves 60 300 roots 60 350 Elettariopsis slahmong leaves 50 400 roots 50 400 Elettariopsis triloba leaves 50 400 roots 50 400 Elettariopsis curtisii leaves 45 400 roots 20 300 Etlingera littoralis leaves 30 300 rhizomes and roots 45 300 Etlingera elatior leaves 40 300 rhizomes and roots 40 300 Etlingera elatior var. Thai queen leaves 15 400 rhizomes and roots 20 350
17
The entire ‘V’ area of the apparatus was filled with distilled water, following which a small volume (5-8 ml) of freshly distilled pentane was added through inlet A (Fig. 2.1). Inlet A was then loosely covered with a piece of aluminium foil to minimize the loss of the essential oil during the hydrodistillation. Ice-cold water was circulated through the condenser for the duration of the hydrodistillation. To minimize condensation of the vapour before it reached the condenser, the left part of the glass apparatus was wrapped with aluminium foil.
Hydrodistillation was carried out for 4 hours; a small volume of pentane was added from time to time through inlet A to ensure that the volume of pentane was maintained at about 5 ml.
At the end of the hydrodistillation, the glass apparatus was left to cool and the essential oil solution in pentane was drained through the stop cock into a separatory funnel where water was removed. The resulting solution was collected into a glass vial (5 ml), concentrated to an appropriate concentration (for GC) by a gentle stream of nitrogen gas, and stored in the refrigerator until required for analysis.
2.3.2 Chromatographic Analysis of the Essential Oils
The essential oils isolated from the various plants were analysed by capillary gas
chromatography (GC) and gas chromatography-mass spectrometry (GC-MS).
2.3.2.1 Gas Chromatography
GC analyses were carried out using a Hitachi G-3000 equipped with a flame ionization
detector (FID). Two fused silica capillary columns of different polarity were employed:
SPB-1 (30 m x 0.25 mm id, film thickness 0.25 μm) and Supelcowax 10 (30 m x 0.25 mm
id, film thickness 0.25 μm). Operating conditions for both these columns were as follows:
initial oven temperature, 50 oC for 1 min, then to 250 oC (for SPB-1) or 230 oC (for
Supelcowax 10) at 4 oC min-1 then held for 10 min; injector and detector temperatures, 275
18
o -1 C; carrier gas, 1.0 ml min N2; split ratio, 50:1; injection volume, 0.4 µL. Peak areas were determined with a Hitachi D2500 Chromato-Integrator. Correction for detector response was not made.
Immediately after each GC analysis of an essential oil, a mixture containing a homologous series of n-alkanes ranging from C5 to C32 was injected into the column under identical
operating conditions. The hydrocarbons were used as standards in the calculation of
retention indices (RI).
For a temperature-programmed GC, the retention index (RI) of a component in the essential
oil was calculated using the following equation (Van den Dool and Kratz, 1963):
⎡ − tt n)( ⎤ = 100iRI ⎢ ⎥ +100n (1) ⎣⎢ + − tt nin )()( ⎦⎥
under the condition that t(n) < t < t(n+i).
t = retention time of the essential oil component.
t(n) = retention time of the alkane with n carbon atoms
which is eluted just before the component.
t(n+i) = retention time of the alkane with (n+i) carbon
atoms which is eluted just after the component.
i = difference in the number of carbon atoms between the
two alkanes.
n = the number of carbon atoms in the alkanes.
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The retention index values obtained based on the calculation using two neighbouring alkanes (i.e. i = 1) give best precision. When i = 1, equation (1) is simplified to:
⎡ − tt n)( ⎤ RI = 100⎢ ⎥ +100n (2) ⎣⎢ n+ − tt n)()1( ⎦⎥
and that was used to calculate the RI values in the present work.
2.3.2.2 Gas Chromatography - Mass Spectrometry
GC-MS analyses were performed using either a ThermoFinnigan GC 2000 coupled to a
Trace MS and equipped with the NIST and MAIN Library softwares, or a Hewlett Packard
5989A GC-MS equipped with the Wiley Library software. The same capillary columns and
GC operating conditions were employed as described in Section 2.3.2.1, the only difference
was the carrier gas used which was helium. Significant operating parameters: ionization
voltage, 70 eV; ion source temperature, 200 oC; scan mass range, 40-350 amu.
2.3.3 Identification of the Essential Oil Components
The components of each essential oil were identified by matching their mass spectra with
those recorded in the MS Library and with those of authentic compounds, if available.
Components were further confirmed by comparison of the experimentally calculated RI
values with those of authentic standards or with values published in the literature.
2.3.4 Determination of Oil Yield
The oil containing pentane was carefully concentrated by a gentle stream of nitrogen gas
until it was free from pentane (indicated by GC analysis) and then weighed. The percentage
yield was determined by dividing the weight of the essential oil with the weight of the fresh
plant material.
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2.4 Isolation and Characterization of the Non-Volatile Constituents in the Leaves,
Fruits, Rhizomes and Roots of Etlingera littoralis
2.4.1 Extraction Procedure
2.4.1.1 Leaves
Air-dried leaves (450 g) were ground into a fine powder and soaked in petroleum ether (4.0
L) at room temperature (28 oC) for a week. The resulting slurry was filtered, and the filtrate was evaporated at 50 oC and under reduced pressure to afford a greenish-black syrup (4.00
g).
2.4.1.2 Fruits
Air-dried fruits (30 g) were ground into a fine powder and soaked in petroleum ether (0.8 L)
for a day at room temperature (28 oC). The resulting slurry was filtered, and the filtrate evaporated at 50 oC to dryness, using a rotarory evaporator, to afford a black syrup (0.35 g).
The residue was next soaked in ethyl acetate (1.5 L) for a day at room temperature. The
slurry was filtered, and the filtrate evaporated to dryness at 50 oC, using a rotarory
evaporator, to give a reddish-brown syrup (1.00 g).
2.4.1.3 Rhizomes and Roots
Air-dried rhizomes and roots (300 g) were finely ground and soaked in petroleum ether (3.0
L) at room temperature (28 oC) for a week, after which the slurry was filtered, the filtrate
evaporated to dryness at 50 oC and under reduced pressure, yielding a reddish-brown syrup
(1.50 g). Next, the residue was soaked in ethyl acetate (4.0 L) at room temperature (28 oC) for two weeks. The slurry was filtered; the filtrate evaporated to dryness at 50 oC and under
reduced pressure to give a brown syrup (2.51 g).
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2.4.2 Separation Techniques
2.4.2.1 Thin Layer Chromatography
Preliminary investigation of the chromatographic separation of the crude extracts was carried out using silica gel TLC (5 cm x 1 cm). Different solvent systems were tried to find one which could achieve the best separation for each of the mixtures. The selected solvent systems were utilized later in the column chromatographic separation of the crude extracts to isolate the components. Developed TLC plates were visualized using an UV lamp (365 nm), or dipped in reagents such as 1% FeCl3 (heating not required), or 5% methanolic
o H2SO4 followed by heating at 100-105 C until full development of colour has occurred to aid visualization (Jork et al., 1990; Harborne, 1998).
2.4.2.2 Column Chromatography
Column chromatography using either silica gel or Sephadex LH-20 was employed for
component isolation or purification. When silica gel was used as the adsorbent, elution was
carried out using either isocratic or gradient solvent systems. However, in the case of
Sephadex LH-20, various proportions of mixtures of methanol : water (v/v) were used.
Eluates were collected in 20 ml fractions and the composition of each fraction was
monitored by TLC. Fractions showing similar TLC profiles were pooled, and the solvents
were evaporated off. Repeated purification using the same technique was carried out until a
pure compound was isolated.
2.4.2.3 Preparative Thin Layer Chromatography
This method was only employed for the final purification of F2 and L2 which could not be
achieved through column chromatography. Each silica gel plate (20 cm x 20 cm x 0.5 mm)
was loaded with 20 mg of sample in a narrow band. The plates were placed in a large
covered glass chamber (30 cm x 30 cm x 10 cm), developed in a suitable isocratic solvent
system and visualized as described in Section 2.4.2.1. The location of a component on the
plate was marked, and the absorbent in the marked region was scrapped off and placed into
22
a conical flask and extracted repeatedly (5 x 20 ml) with chloroform. The component was recovered on evaporation of the chloroform (Zubrick, 1992).
2.4.3 Isolation and Purification
2.4.3.1 Leaves
2.4.3.1.1 Petroleum Ether Extract
The petroleum ether extract (4.00 g) (section 2.4.1.1) was chromatographed on a silica gel column (100 g). Elution was carried out using mixtures of petroleum ether : ethyl acetate in proportions of 80:20 (v/v) (300 ml), 40:60 (v/v) (300 ml) and 0:100 (v/v) (200 ml) sequentially to afford fractions LA (2.00 g), LB (0.30 g) and LC (0.80 g), respectively. LA
was fractionated on a silica gel column (100 g), eluted successfully using mixtures of
petroleum ether : chloroform in proportions of 100:0 (v/v) (300 ml), 80:20 (v/v) (300 ml)
and 60:40 (v/v) (200 ml), followed by mixtures of chloroform : ethyl acetate in ratios of
80:20 (v/v) (200 ml) and 40:60 (v/v) (200 ml), to afford sub-fractions LA 1 (0.05 g), LA 2
(0.02 g), LA 3 (1.30 g), LA 4 (0.01 g) and LA 5 (0.02 g), respectively. Column
chromatography of LA 3 over silica gel (30 g) with successive elutions using mixtures of
petroleum ether : ethyl acetate in proportions of 90:10 (v/v) (150 ml) and 70:30 (v/v) (150
ml) afforded sub-fractions LA 3.1(0.60 g) and LA 3.2 (0.40 g), respectively. LA 3.1 was further separated by a 20 g silica gel column with petroleum ether : chloroform as the eluting solvent (75:25) (v/v) (300 ml) to give LA 3.1.1 (0.32 g) and LA 3.1.2 (0.18 g).
Column chromatography of LA 3.1.1 over a 20 g silica gel column using an isocratic solvent system of petroleum ether : ethyl acetate (90:10) (v/v) (200 ml) provided LA 3.1.1.1
(0.10 g), LA 3.1.1.2 (0.10 g) and L1 (30 mg) (Appendix A1). L1, a colourless oil, [α]D +
o 0.40 (CHCl3, c 2.0) gave a single brown spot with 5% H2SO4 on TLC [petroleum ether :
ethyl acetate (95:5), Rf = 0.40; petroleum ether : acetone (95:5), Rf = 0.34; petroleum ether : chloroform (50:50), Rf = 0.66; petroleum ether : diethyl ether (90:10), Rf = 0.40; petroleum ether : chloroform : ethyl acetate (70:25:5), Rf = 0.63; petroleum ether : chloroform : acetone
(80:15:5), Rf = 0.60]. Final purification of LA 3.1.1.2 to yield L2 (25 mg) was achieved by
23
chromatography over a silica gel column (15 g) with petroleum ether : chloroform (70:30)
(v/v) (100 ml) as the eluting solvent, followed by preparative TLC using the same solvent
o system (Appendix A1). L2, isolated as a yellow oil [α]D -14.4 (CHCl3, c 1.7), gave a single
red spot with 5% H2SO4 on TLC [petroleum ether : ethyl acetate (95:5), Rf = 0.71;
petroleum ether : acetone (98:2), Rf = 0.66; petroleum ether : chloroform (70:30), Rf = 0.63; petroleum ether : diethyl ether (95:5), Rf = 0.63; petroleum ether : chloroform : ethyl acetate
(90:5:5), Rf = 0.69; petroleum ether : chloroform : acetone (95:3:2), Rf = 0.66].
2.4.3.2 Fruits
2.4.3.2.1 Ethyl Acetate Extract
The ethyl acetate extract (1.00 g) (section 2.4.1.2) was column chromatographed over
Sephadex LH-20 (25 g), using methanol (300 ml) as the eluting solvent, to afford fractions
FA (0.30 g) and FB (0.50 g). FB was further fractionated by passing it through a 25 g silica
gel column, eluting with mixtures of chloroform : methanol in proportions of 80:20 (v/v)
(200 ml), 70:30 (v/v) (300 ml), 60:40 (v/v) (300 ml) and 50:50 (v/v) (200 ml) successively
to afford sub-fractions FB 1 (0.05 g), FB 2 (0.20 g), FB 3 (0.10 g) and FB 4 (0.05 g),
respectively. Final purification of FB 3 to yield F1 (48 mg) was achieved through a small
Sephadex LH-20 column (5 g), using 30 ml methanol : water (50:50) (v/v) as the eluting
solvent (Appendix A2). F1 was crystallized as white needles from ethanol, mp 171 – 173 o o C, [α]D – 13.9 (MeOH, c 3.2). F1 gave a single grayish black spot with 1% FeCl3, and a reddish orange spot with 5% H2SO4 on TLC [chloroform : ethyl acetate (50:50), Rf = 0.11;
ethyl acetate, Rf = 0.69; chloroform : acetone (50:50), Rf = 0.43; chloroform : ethyl acetate : acetone (1:1:1), Rf = 0.40; chloroform : methanol (80:20), Rf = 0.37].
2.4.3.2.2 Petroleum Ether Extract
The petroleum ether extract (0.35 g) (section 2.4.1.2) was chromatographed on a 20 g silica gel column, eluting using mixtures of petroleum ether : ethyl acetate in proportions of 100:0
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